Encapsulation of Fibrinogen With Calcium Carbonate for Hemorrhage Control

Combat-related hemorrhage remains the leading cause of preventable death, accounting for over 60% of fatalities. Dressings containing thrombin-calcium carbonate (CaCO3) particles combined with protonated tranexamic acid have been shown to reduce blood loss and mortality in severe bleeding cases. The integration of fibrinogen further enhances hemostatic efficacy. This study explores the preparation of fibrinogen-encapsulated CaCO3 particles using three innovative methods and evaluates their hemostatic and self-propelling properties.

Fibrinogen-CaCO3 particles were synthesized via water-oil-water encapsulation, precipitation, and gas diffusion methods. For the encapsulation method, fibrinogen in 20 mM HEPES and 1 M carbonate solution was emulsified with oil, Tween 80, and Span 80, and then added to a calcium chloride (CaCl2) solution, and stirred at 400 rotations per minute (RPM) for 10 min. For the precipitation method, fibrinogen was dissolved in 10 mM HEPES and 0.33 M Na2CO3, then combined with 0.33 M CaCl2, and stirred at 200 RPM for 2 h. Alternately, Na2CO3 was replaced with NaHCO3 or (NH4)2CO3. The gas diffusion method involved dissolving fibrinogen in CaCl2 solution, which was exposed to CO2 and NH3 generated from (NH4)2CO3 for 24 h in a desiccator. To quantify fibrinogen distribution, fluorescein isothiocyanate labeling of fibrinogen was performed before particle preparation. Particles were collected, washed, lyophilized, and characterized using microscopy, rotational thromboelastometry, and video motion tracking.

The method and preparation conditions significantly influenced the properties of fibrinogen-CaCO3 particles. Both precipitation and encapsulation methods produced spherical micrometer-sized particles, while the gas diffusion method resulted in irregular shaped particles. Variations in carbonate sources affected particle yield and size. Fibrinogen inclusion led to larger particles in the encapsulation method. Fluorescent microscopy confirmed the successful encapsulation of fibrinogen in all methods, with precipitation particles showing the strongest hemostatic effect. All fibrinogen-containing particles exhibited self-propulsion capabilities, with encapsulation particles outperforming others in terms of response time and propulsion speed.

This study demonstrates that multiple techniques can be used to effectively produce fibrinogen-encapsulated CaCO3 particles with hemostatic and self-propelling properties. Further optimization of the formulation is planned to enhance these properties for hemorrhage control.